Our main objective is to develop methods to protect against streptococcal infections in particular and gram-positive infections in general. Group A streptococcal-associated disease (pharyngitis, rheumatic fever, necrotizing fasciitis, impetigo, and scarlet fever, to name a few) affects millions of people, particularly children, worldwide. In developing countries, rheumatic heart disease is the major cause of heart damage in the school-age population. The severity of streptococcal diseases provides the impetus to develop novel methods to both prevent and treat these infections. Using a mucosal vaccine approach we have been successful in achieving cross-protection against heterologous streptococcal serotypes in a mouse model using the conserved region of the streptococcal M protein as the antigen. This approach circumvents the need to prepare type- specific antigens directed to the >120 streptococcal M serotypes. Furthermore, since the only reservoir for group A streptococci is the human pharynx, we have used a model system to successfully decolonized the oral cavity of mice of colonizing group A streptococci using a specific phage lytic enzyme. We hope to use this approach to reduce the bacterial burden in the human population and thus reduce disease. The experiments outlined in this proposal will expand these findings to develop both a new generation streptococcal vaccine and a prophylactic treatment to prevent streptococcal infections. In addition, during our studies, we identified a novel glycosylated enzyme in group A streptococci that is responsible for both attachment of surface proteins and cell wall assembly. This novel molecule is not ribosomally synthesized and as such we wish to determine its method of assembly. Since this glycoprotein is also found in other gram- positive bacteria (i.e., staphylococci and pneumococci) an understanding of its biosynthesis should offer novel targets for antibiotic development against these and perhaps other bacterial pathogens. The complete or partial M protein has not yet been crystallized. To better understand the details of the M protein structure and its multifunctional capacity, we will collaborate to solve the crystal structure of the biologically active N- and C-terminal halves of the molecule. Since many surface proteins on gram-positive bacteria are extended coiled-coil structures of modular design, structural data for the M molecule will allow for a better understanding of other surface proteins on gram-positives. In summary, our continued studies on gram-positive pathogenic bacteria over the >30 years of this grant has revealed new information regarding these pathogens enabling new strategies for their control;this application will continue in this direction.